ARTICLE
A Nanoengineering Approach for
Investigation and Regulation of Protein
Immobilization
Yih Horng Tan, Maozi Liu, Birte Nolting, Joan G. Go, Jacquelyn Gervay-Hague, and Gang-yu Liu*
Department of Chemistry, University of California, Davis, California 95616
ABSTRACT It is known that protein attachment to surfaces depends sensitively upon the local structure and
environment of the binding sites at the nanometer scale. Using nanografting and reversal nanografting, both
atomic force microscopy (AFM)-based lithography techniques, protein binding sites with well-defined local
environments are designed and engineered with nanometer precision. Three proteins, goat antibiotin
immunoglobulin G (IgG), lysozyme, and rabbit immunoglobulin G, are immobilized onto these engineered
surfaces. Strong dependence on the dimension and spatial distribution of protein binding sites are revealed in
antibody recognition, covalent attachment via primary amine residues and surface-bound aldehyde groups. This
investigation indicates that AFM-based nanolithography enables the production of protein nanostructures, and
more importantly, proteinⴚsurface interactions at a molecular level can be regulated by changing the binding
domains and their local environment at nanometer scale.
KEYWORDS: nanografting · reversal nanografting · arrays of nanostructures ·
atomic force microscopy · self-assembled monolayers · polyvalent interactions ·
multipoint protein binding · protein immobilization · local structures
M
*Address correspondence to
[email protected].
Received for review August 8, 2008
and accepted October 06, 2008.
Published online October 29, 2008.
10.1021/nn800508f CCC: $40.75
© 2008 American Chemical Society
2374
uch effort has been devoted recently to protein immobilization
on surfaces because surfacebound proteins have important applications in drug screening, biosensing, bioassaying, and protein characterization.1
Fundamental interactions between proteins and solid surfaces include one or a
combination of physical adsorption,2⫺7
electrostatic forces,8 specific
recognition,9⫺11 and covalent
binding.3,6,12⫺14 These interactions are
found to depend sensitively upon the local
structures and environment of protein binding sites on surfaces.15,16 Much work has
been devoted to surface modification for
protein adhesion, which has been reviewed
and discussed extensively.15,17⫺19 For example, the coverage and overall morphology of physically adsorbed bovine serum albumin (BSA) on mixed self-assembled
monolayers (SAMs) depend upon the surface structure of the monolayer (e.g., phase
segregation or lateral heterogeneity).20 Using a bilayer platform, Yang et al. reported
VOL. 2 ▪ NO. 11 ▪ TAN ET AL.
that a higher binding affinity was observed
for anti-dinitrophenyl-keyhole limpet
hemocyanin IgG antibodies to high density
hapten-containing membranes13,21 in comparison to the monovalent binding of antidinitrophenyl-keyhole limpet hemocyanin
IgG antibodies to hapten ligand. The apparent dissociation constant, KDapp, for the
bivalent binding of an antibody to the hapten ligand decreased by about a factor of
10 as the ligand density increased.13,21 Similar dependence of surface heterogeneity at
the nanometer level was also demonstrated
by Ostuni et al.4 In their study, they found
that the coverage of the adsorbed proteins
via the interaction of the protein’s hydrophobic groups with hydrophobic functional
terminated molecules in a mixed SAM system increases as a function of the trityl terminal group’s physical size (⫺CH2Ph ⬍
⫺CHPh2 ⬍ ⫺CPh3).4 Theoretical approaches, such as the Temkin model and
stoichiometric displacements model, have
also been reported to deal with proteins’
strong binding affinity due to multiple interactions between functional groups of the
protein and the corresponding binding sites
on surfaces.22⫺24
Prior approaches to regulating surface
heterogeneity to affect protein immobilization mainly relied on a mixing-and-growth
method due to its simplicity.22 By regulating
the composition of protein binding components and surface reaction conditions, this
method was proven to be effective in
changing surface domain structures and
therefore impacted protein adhesion.2,25
To attain a higher degree of control of
protein⫺surface interactions, instead of relying on the tradeoff of thermal dynamics
and kinetics of surface reactions in the
mixing-and-growth, AFM-based nanolithogwww.acsnano.org
ARTICLE
raphy techniques, such as nanografting,25⫺27 were
used in this investigation. AFM lithography is best
known for the production of nanostructures of
ligands, DNA, and proteins on surfaces.4,28⫺30 To
further take advantage of nanolithography, this
work focuses on producing designed nanodomains of protein binding sites, with the precision of a single protein molecular size or
smaller, and then characterizing protein molecules upon their interactions with these engineered surfaces in situ. The regulation of protein
attachment to these engineered surfaces is
clearly demonstrated.
RESULTS AND DISCUSSION
Nanografting and Reversal Nanografting. The concept
and procedure of nanografting has been extensively discussed in our previous publications.26,27,31
The key steps of nanografting, that is, imaging,
shaving-and-replacement, and imaging again, are
schematically shown in Figure 1. The matrix SAM is
formed via natural growth, while thiol containing
protein binding termini (or designed mixture) is in
solution phase and is attached to the Au surface following the shaving trajectory of the AFM tip. For
the investigation of protein adsorption, nanograftFigure 1. Nanografting and reversal nanografting process to reveal the key
ing provides the simplest and very effective means fabrication steps.
for precise engineering of nanostructures with a
designed single component26 or with mixed comarray production. Modifications to nanografting are
ponents at controlled heterogeneity.25 In the case of
made to overcome those limitations. The new and
protein attachment via covalent binding between its
modified nanofabrication method, referred to as reprimary amine groups and aldehyde termini on the subversal nanografting, is illustrated in Figure 1. Similar
strate, a binary SAM of
to nanografting, this new method also has three
hexanethiol (referred to as C6) and 11-mercapto-1main steps of imaging, shaving-and-replacement,
undecanal disulfide [⫺S(CH2)10CHO]2 (referred to as
and imaging again. In contrast to nanografting, the
C10CHO due to the cleavage of the disulfide bond on
matrix SAM contains the protein binding termini,
32⫺34
gold) was used.
For binary SAMs, the degree of
while the inert thiols are in the solution. During the
phase segregation can be regulated by varying the fabshaving-and-replacement steps, these protein non25
rication parameters, such as the shaving speed. The
lateral heterogeneity ranged from near molecular level adherent thiols can be placed at a designed location
mixing to segregated nanodomains with different sizes to form boundaries between reactive thiols, and
therefore, large sized protein binding structures can
and separations. The size and distribution of aldehyde
be divided into small sized nanostructures. By condomains in these nanostructures, therefore, can be
trolling the shaving size and the spacing between
regulated by varying the nanografting parameters to
match the dimension of protein and the primary amine the shaving lines, arrays of nanostructures with degroups on individual protein surfaces for the investiga- sired size and numbers can be fabricated in a short
period of time.
tion of covalent immobilization. Similar concepts may
In terms of production of nanostructure arrays, rebe applied to study protein adsorption via electrostatic
versal nanografting has the advantages of high fidelity
van der Waals or hydrophobic interactions.
to the design, excellent uniformity of the array eleFor the investigation of specific interactions, such
ments, minimal subject to thermal drifts, and highly imas biorecognition, many arrays of nanostructures with
proved throughput. Reversal nanografting also allows
designed feature sizes and separations must be profor flexibility to select the inert component(s) to reach
duced. Nanografting is a serial process in nature prehigh self-assembly speed and to avoid or minimize the
senting limitations such as vulnerability to thermal
exchange reactions and/or to attain the designed local
drift to maintain the designed geometry, subject to
exchange due to prolonged soaking in thiol solution environment (e.g., hydrophobic vs hydrophilic) surrounding the nanostructures.
and the possibility of tip wear are the obstacles for
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VOL. 2 ▪ NO. 11 ▪ 2374–2384 ▪ 2008
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ARTICLE
Figure 2. Structure of immunoglobulin G and lysozyme. (A)
Y-Shaped immunoglobulin G molecule structure containing
the 83 lysine groups (highlighted in red) and composed of
two antigen binding fragments, Fab (highlighted in blue). (B)
Lysozyme molecule with the 13 lysine groups highlighted
in red. The molecular structures were rendered by Chimera
from the Protein Data Bank.
Production of Three Arrays of Biotin Nanostructures Using Reversal
Nanografting. The design of arrays of biotin nanostructures took the protein structure and antibody recognition reaction into consideration. The structure of an IgG
was obtained from the protein data bank (ID code 1IGT)
and is shown in Figure 2A to highlight the surface binding sites. Molecular graphics images were produced using the University of California, San Francisco (UCSF)
Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the UCSF (supported
by NIH P41 RR-01081).35 The Y-shaped IgG is composed
of two antigen-binding fragments, Fab (highlight in
blue color in Figure 2A), and a crystallizable fragment.
Both fragments are heavily decorated with lysine
groups. Two important features are (a) the antigen
binding sites (i.e., the Fab domains), for specific binding of IgG to surfaces, and (b) all lysine residues (highlighted in red in Figure 2), for covalent immobilization
onto CHO termini of SAMs. The typical dimensions of
IgG are approximately 14.5 nm ⫻ 8.5 nm ⫻ 4.0 nm, with
antigen binding sites separated by 13.7 nm.36 There
are approximately 83 lysine groups per IgG.
Figure 3A shows three arrays of biotin nanostructures fabricated using reversal nanografting. The three
2376
VOL. 2 ▪ NO. 11 ▪ TAN ET AL.
arrays represent three characteristic scenarios for the
engineered biotin nanostructures: (i) nanostructures
whose domain size is smaller than the goat antibiotin
IgG and the separation between nearest neighbors is
also smaller than the Fab separation; (ii) nanostructures
whose domain size is smaller while the separation between nearest neighbors matches the separation of two
Fab domains in IgG; and (iii) nanostructures whose domain size is larger than IgG and separations match the
separation of two Fab domains. A biotin-terminated
SAM was formed by 48 h immersion of a bare gold thin
film in a N-biotinyl-N=-(11-mercaptoundecyl)-3,6dioxaoctane-1,8-diamine solution. The inert spacer molecules, hexanethiol, were nanografted into the biotin
SAM, using reversal nanografting. Three nanostructures
referred to as 1, 2, and 3 cover the total areas of 450
nm ⫻ 500 nm, 470 nm ⫻ 500, and 410 nm ⫻ 1065 nm,
respectively, as shown in Figure 3A. The zoom-in images, 300 nm ⫻ 300 nm area, of the three arrays are
shown in panel D, E, and F, respectively, from which the
dimension of the nanostructures can be quantified
(see Table 1).
Array 1, shown in Figure 3D, contains a 33 ⫻ 33 biotin nanoarray, with square features of 5.2 nm ⫻ 5.2 nm.
The separation of each element from edge-to-edge is
4.3 ⫾ 0.6 nm. The center-to-center separation among
each nearest element is 9.2 ⫾ 0.6 nm. The dimension of
individual nanosquares is too small for the Fab to form
bivalent binding. Among collisions of IgG with the nearest neighboring biotin nanosquares, most would not result in bivalent binding because the separation is too
small, except in the extreme case where Fab collides
with two far edges of the neighboring nanosquares.
The probability of matched collisions is very slim, thus
the coverage of IgG on array 1 is likely to be very low.
Array 2, as shown is Figure 3E, has 16 ⫻ 18 biotinterminated nanostructures, with a feature size of 12.7
nm ⫻ 12.7 nm. The edge-to-edge separation distance
is 5.9 ⫾ 0.7 nm, and center-to-center separation is increased to 18.0 ⫾ 1.2 nm. Array 2 provides excellent geometry matching to the Fab domains of the IgG (i.e.,
13.7 nm). Any collisions of Fab with the nearest neighbor nanosquares would satisfy the geometry required
for bivalent recognition. The biorecognition occurs by
bridging to the nanosquares. The third array has 8 ⫻ 18
elements, with rectangular features of 10.3 nm ⫻ 31.9
nm. Along the x-axis (long axis of each nanofeature), the
separation distance between the neighboring biotin
nanofeatures is 8.2 ⫾ 1.9 and 40.2 ⫾ 1.9 nm from edgeto-edge and center-to-center, respectively. Similarly,
for the y-axis (short axis of each nanofeature), the separation distance between the neighboring biotin structures from edge-to-edge is 5.5 ⫾ 0.7 nm. The center-tocenter separation distance is 16.3 ⫾ 0.7 nm. The
geometry of array 3 allows biorecognition to occur
within each feature or by bridging nearest neighbor
nanostructures of biotin along various azimuthal direcwww.acsnano.org
ARTICLE
Figure 3. Antibiotin IgG reacts with thiolated biotin nanostructure arrays fabricated using reversal nanografting. (A) A 1500
nm ⴛ 1500 nm area revealing three different areas of nanoarrays of biotin produced using reversal nanografting as indicated
by 1, 2, and 3. (B) The same area as (A) after antibiotin IgG injection. (C) A combined cursor plot as defined by the line in
(A) and (B) is shown in order to reveal the local height change before and after exposure to the IgG solution. (D) A 300 nm
ⴛ 300 nm zoom-in AFM topographic image of area 1. There are 1089 biotin nanofeatures with feature size of 5.2 nm ⴛ 5.2
nm. (E) A 300 nm ⴛ 300 nm zoom-in AFM topographic image of area 2. There are 288 nanofeatures with dimension of 12.7
nm ⴛ 12.7 nm. (F) A 300 nm ⴛ 300 nm zoom-in AFM topographic image of area 3. There are 144 nanofeatures with dimension of 10.3 nm ⴛ 31.9 nm.
tions. The differences between engineered nanostructures and the nanodomains in matrix SAMs are significant both qualitatively, square and rectangle domains
versus ellipses, as well as quantitatively as summarized
in Table 1.
Antibody Recognition of Biotin in the Presentation of SAMs
and Nanostructures. The antigen (i.e., biotin termini)
present in three arrays of nanostructures and the surrounding SAM were exposed to the same goat antibiotin IgG solution, 10 ␮g/mL in 1⫻ PBS buffer (pH 7.0),
and were allowed to incubate for 30 min. Upon wash-
ing with 1% Tween 20, the surface was then imaged in
PBS and is shown in Figure 3B. The presentation of
biotin clearly impacted the subsequent protein adsorption, comparing Figure 3A with 3B. First, the coverage
of IgG varies depending on the local environment of antigens. Only minute amounts of IgG, 15 ⫾ 2 molecules,
were seen in array 1, from Figure 3B, while more antibodies are found on nanostructures 2 and 3, 137 ⫾ 5
and 155 ⫾ 20, respectively. In the biotin SAM region in
Figure 3B, the number of adsorbed protein is 607 ⫾ 40.
The number of proteins was counted using the follow-
TABLE 1. Antibody Recognition of Surface-Bound Biotin under Four Different Presentations
biotin
SAMs
matrix
array “1”
array “2”
array “3”
biotin nanostructure
preparation
total area
(nm ⴛ nm)
total number
of features
feature size
(nm ⴛ nm)
natural
growth
reversal
nanografting
reversal
nanografting
entire
Surface
450 ⫻ 500
nanodomains covering
entire surface
3969
470 ⫻ 500
reversal
nanografting
410 ⫻ 1065
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separation
(nm)
edge-to-edge
center-to-center
matching to
Fab sites
13.2 ⫻ 7.0
(domain size)
5.2 ⫻ 5.2
4.8
10.9
excellent
4.3
9.2
poor
961
12.7 ⫻ 12.7
5.9
18.0
465
10.3 ⫻ 31.9
8.2
40.2
5.5
16.3
excellent among
nearest neighbor
biotin nanosquares
excellent within each and
among nearest neighbor
biotin nanostructures
VOL. 2 ▪ NO. 11 ▪ 2374–2384 ▪ 2008
protein
coverage
(protein/␮m2)
450 ⫾ 29
67 ⫾ 9
582 ⫾ 22
355 ⫾ 45
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The observations above indicate that antibody recognition
can be regulated by varying the
geometry, allowing the availability in size and distance matching
of surface-bound antigens, using reversal nanografting. This
conclusion, based on the correlation between the structure of
SAMs and three arrays of biotin
nanostructures and the protein
attachment, is illustrated schematically in Figure 4. Using the
biotin SAM matrix as a reference, where biotin molecules
are readily available for goat antibiotin IgG to attach, randomly
orientated IgG were observed, as
illustrated in Figure 4D. Nanoengineered areas reveal different outcomes. Figure 4A represents nanostructures such as array 1, whose elemental size is too
small to accommodate one antibody binding. Since the distance
between neighboring features
(9.2 nm) does not match the
geometric separation of two Fab
binding domains (13.7 nm), the
Figure 4. Antibiotin IgG (top view and side view) reacted with biotin nanofeatures at different scenarios: (A) feature size and separation of biotin, poorly match the goat-antibiotin IgG Fab domain; (B) probability for goat antibiotin
feature size is too small while the separation between nearest neighbor nanostructure is excellent in
IgG bivalently binding to neighmatching the geometric separation of two Fab domains of IgG; and (C) the individual biotin nanofeaboring elements is also poor. As
tures provide an excellent match to the Fab domain, and excellent matching of the Fab domain to
biotin also achieved among nearest neighbor biotin nanostructures. (D) IgG reacts among the neara result, little protein attachment
est neighbor biotin domain.
is observed for nanostructures
in array 1.
ing dimensions as the threshold: 20 nm lateral widths
Figure 4B represents nanostructures (e.g., array 2 in
and 5.8 nm in AFM apparent height, based upon indiFigure 3A) whose feature size is too small for divalent anvidual IgG molecules imaged under AFM. This threshtibody binding. However, the geometry among nearest
old is determined from protein molecules in the low
neighbor nanofeatures matches the Fab binding to allow
coverage region and is consistent with previous AFM in- antibody recognition. Such geometry matching resulted
vestigations.12 For comparison, the protein coverage
in effective antibody recognition, as shown in Figure 3B
was quantified using the number of IgG molecules per
and illustrated in Figure 4B. Even though array 2 was not
␮m2: 67 ⫾ 9, 582 ⫾ 22, 355 ⫾ 45, and 450 ⫾ 29 IgG
designed to align the proteins, the immobilized protein
molecules per ␮m2 for arrays 1, 2, 3, and matrix region, layer exhibits a higher degree of homogeneous orientarespectively. The morphology of the IgG molecules in
tion in comparison to the randomly placed IgG onto the
array 2 differs from the rest by exhibiting brighter con- biotin SAM in the surrounding matrix. To demonstrate the
trast, as shown in Figure 3B. The local height before and robustness of this regulation, array 3 was designed to alafter exposure to the protein solution is shown in Figlow IgG to attach within each nanofeature and to bridge
ure 3C, where a combined cursor profile reveals clearly neighboring nanostructures along various azimuthal dithat IgG molecules on nanostructure 2 appear taller
rections. In other words, array 3 is similar to the matrix
than that in the matrix region. This observation is conSAM. As a result, the antibody recognition occurred in a
sistent with the explanation that protein molecules on
similar fashion as on biotin SAMs, as revealed in Figure 3B
array 2 have a higher coverage and share similar lateral and illustrated in Figure 4C.
orientations in comparison to other regions. The higher
The concept of regulations via nanolithography
degree of coverage and homogeneity manifests into
may be extended to the forward phase protein microarless deformation under the AFM tip
rays, where the specific orientation of the IgG molecules can be obtained by using Fc binding functionalduring contact imaging.
VOL. 2 ▪ NO. 11 ▪ TAN ET AL.
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ARTICLE
ities, such as protein A or G. Protein A or G can
be immobilized on CHO-terminated SAM nanostructures. This attachment enables Fab fragments to face the solution for binding specific antigens. In dealing with a crude mixture protein,
this methodology would also be useful by production of multiple arrays, each with functionality
specific to targeted proteins, such as RGDterminated nanostructures for integrin, and IgG
nanostructures specific to F-actin, etc. This is
analogous to the concept of multiplexing, except
that nanografting enables nanoscale control. In
situ characterization with resolution of individual
proteins is also a benefit to verify adsorption and
to determine protein coverage and orientation in
situ.
Covalent Immobilization of IgG on Aldehyde Domains
Presented in Naturally Grown and Nanografted Binary SAMs.
Previously, we have demonstrated that nanografting can be utilized to regulate the lateral heterogeneity of binary SAMs, using a mixture of
1-hexanethiol and 1-octadecanethiol.25 In this investigation, we extended the regulatory capability
to binary SAMs with protein adhesive groups,
Figure 5. Binary mixed SAMs of hexanethiol and C10CHO with a ratio of 1:1, total
such as aldehydes. Figure 5A is a 500 nm ⫻ 500
concentration 0.1 mM, formed from natural growth and nanografting. (A) AFM tonm AFM topographic image revealing two nanopographic image of the mixed SAM formed by co-adsorption of C6 and C10CHO
grafted rectangular patterns, at 180 nm ⫻ 120 nm from ethanol solution with mixed SAMs formed using the nanografting method.
(B) High-resolution 100 nm ⴛ 100 nm image of the mixed SAM formed from natuand 220 nm ⫻ 205 nm, labeled as pattern 4 and
ral growth. (C) A 100 nm ⴛ 100 nm nanostructure produced by nanografting
5, within the mixed SAM of C6 and C10CHO. The
from the mixed thiol solution, C10CHO/C6 (0.1 mM 1:1, using fabrication rate at
matrix SAM clearly exhibits phase segregation, as 100 nm/s). (D) AFM topographic image of the same area after injection of 10
expected for SAMs formed from co-adsorption of ␮g/mL rabbit IgG in 10 mM HEPES buffer (pH 6.8) for 30 min. AFM topographic image of the same area, after the surface was thoroughly washed with 1% Tween
C6 and C10CHO in solution.25 The degree of het20 to remove noncovalently bound proteins.
erogeneity in the two nanostructures (4 and 5) is
Upon incubation in a rabbit IgG solution, 10 ␮g/mL
less than the matrix by comparison, as shown in Figure
in 10 mM HEPES buffer (pH 6.8), for 30 min, the surface
5A. Quantitative comparison may be extracted from
was washed with 1% Tween 20 and then imaged in
zoom-in scans (100 nm ⫻ 100 nm) as shown in Figure
HEPES medium. As shown in the 500 nm ⫻ 500 nm to5B,C for the matrix and nanostructures, respectively.
pographical image in Figure 5D, the protein adsorption
Each C10CHO domain in the matrix region adopts irvaries based on the local structure of aldehyde doregular and elongated shapes with a typical domain
mains. The lateral width of 20 nm and 1.5 nm in AFM apsize ranging from 8.4 to 19.7 nm on the long axis and
parent height was used as the threshold and guide for
from 4.7 to 10.8 nm on the short axis. The C10CHO dothe immobilized IgG. There are approximately 580 ⫾ 53,
main size in nanostructure 4 is less irregular, with 7.1
27 ⫾ 3, and 25 ⫾ 4 IgG molecules in the matrix area
nm ⫻ 5.5 nm in dimension, and the distance between
and nanostructures 4 and 5, respectively. After normalthe neighboring C10CHO domains from center-toization, the surface coverage of IgG on the surface of
center and edge-to-edge is 12.1 and 6.5 nm, respec2
tively. The quantitative information of the domain struc- the naturally grown region is 2213 ⫾ 212 proteins/␮m ,
which is almost two times the coverage on nanoengitures is summarized in Table 2.
TABLE 2. Lateral Heterogeneity of Binary Component SAMs Prepared by Natural Self-Assembly and Nanografting
domain separation (nm)
protein coverage
mixed SAMs
SAM preparation
typical domain size (nm ⴛ nm)
center-to-center
edge-to-edge
C10CHO/C6 (Matrix)
array “4”
array “5”
C10CHO/C6 (Matrix)
array “6”
natural growth
nanografting
nanografting
natural growth
nanografting
12.6 ⫻ 7.4
7.1 ⫻ 5.5
8.4 ⫻ 6.7
15.8 ⫻ 10.9
9.3 ⫻ 4.6
14.4
12.1
10.1
20.1
8.6
5.1
6.5
5.0
8.4
3.9
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(protein/␮m2)
2,213 ⫾ 212
1,184 ⫾ 138
625 ⫾ 89
1,923 ⫾ 150
1,883 ⫾ 111
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ografted regions, where the domains
are 30% smaller (7 nm) and the separation (12 nm) is not well matched to
the amine residues in the Fab domain,
as illustrated in Figure 6.
Covalent Immobilization of LYZ on Aldehyde
Domains Presented in Naturally Grown and
Nanografted Binary SAMs. To demonstrate
the generic concept of regulating
protein⫺surface interactions, a
smaller protein, LYZ, was used. The
SAM matrix is a mixture of C6 and
C10CHO and was formed by soaking
gold films into a 0.02 mM thiol solution with C10CHO/C6 ⫽ 1:1. Figure 7A
is an AFM topograph of 500 nm ⫻
500 nm with a 190 nm ⫻ 190 nm nanografted rectangular nanostructure 6
within. Zooming into both regions,
the 100 nm ⫻ 100 nm scans shown
in Figures 7B,C clearly reveal the difference of the phase segregation in
the mixed SAM and the nanografted
area. The bright contrast represents
the C10CHO domains, and the dark areas are the C6 domains in the topographic images. Most of the C10CHO
domains in the matrix are 15.8 nm ⫻
10.9 nm in size, with center-to-center
Figure 6. Schematic diagram of rabbit IgG and lysozyme immobilization on binary mixed SAMs and edge-to-edge separations around
of hexanethiol and C10CHO at final ratio and concentration of 1:1, 0.1 and 0.02 mM, respectively,
20.1 and 8.4 nm, respectively. In the
formed from natural growth and nanografting. The top view and side view of the immobilized
nanografted region shown in Figure
IgG and lysozyme on the aldehyde-terminated nanostructure domain, both in natural growth
and nanografted region, are presented.
7, the aldehyde domains are smaller,
9.3 nm ⫻ 4.6 nm, with the center-toneered structure 4 (1184 ⫾ 138 protein/␮m2) and four
center and edge-to-edge separations of 8.6 and 3.9
times the coverage as nanostructure 5 (625 ⫾ 89 pronm, respectively. Quantification of the domains is sumtein/␮m2). This observation may be rationalized by the marized in Table 2.
nature of protein immobilization onto aldehyde termini,
Figure 7D is the AFM topographic image showing
that is, forming multiple covalent imine bonds with pri- the same area after exposing the surface to a LYZ solumary amine residues at the interface. All 83 lysine resition of 5 ␮g/mL in 10 mM HEPES buffer (pH 6.8). Bright
dues are highlighted as red to reveal their distributions spots shown in Figure 7D correspond to LYZ attachwithin the IgG molecules (Figure 2). Assuming equal re- ment to SAM. The coverage in the matrix region at 1923
activity, the SAMs with CHO domains equal to or larger ⫾ 150 LYZ/␮m2 is almost equal to that in the nanothan the dimension of IgG (14.5 nm ⫻ 8.5 nm ⫻ 4.0
grafted region, 1883 ⫾ 111 LYZ/␮m2.
nm) would have higher probability to form multipoint
This observation may be rationalized by the size of
bonds than small domains. In addition, the amine group LYZ and distribution of amine residues in the protein. As
at the Fab domain of IgG has a 50% higher reactivity
shown in Figure 2B, LYZ is ellipsoidal in shape and smaller
than the Fc domain toward aldehyde groups accordthan IgG, with dimensions of 4.5 nm ⫻ 3.0 nm ⫻ 3.0 nm.
ing to the study by Hara et al.,37 suggesting that the
The 13 lysine groups are highlighted in red in Figure 2B to
availability of CHO at 14.5 nm would facilitate the Fab
reveal the prospects for CHO attachment. Since the indibridging formation on surface aldehyde. Such availabil- vidual domains in the matrix and nanografted regions exity can be provided by large (⬎14.5 nm) domains or
ceed the size of LYZ, similar coverage, assuming multiby small domains with the desired separations. Taking
point covalent bond formation between protein and CHO
both factors into consideration, the aldehyde domains
domains, is expected and is illustrated in Figure 6. For
in the matrix (shown in Figure 5B) exhibit a more optiregulating LYZ attachment, smaller nanofeatures than
mized lateral size (10 nm) and separation (14.4 ⫾ 1.2
the present work must be designed and produced. Therenm) for IgG immobilization than that of the nanfore, the design and fabrication of SAM nanostructures
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CONCLUSION
Using nanografting and reversal nanografting, we have demonstrated that nanostructures of protein binding termini can be produced with high spatial precision and with
sufficient throughput for the investigation of
subsequent protein attachment. The coverage
as well as orientation of protein molecules can
be regulated, to a large degree, by changing
the dimension and separation of each nanoelement, in the case of biotin and antibiotin IgG
reactions, and by changing the nanografting
conditions, in the case of covalent immobilizations. The approach reported in this work is of
generic importance because it provides alternative means to regulate protein immobilization on surfaces. The variation of protein coverage indicates the degree of control that this
approach enables, which should be beneficial
Figure 7. LYZ protein molecules covalently immobilized onto C10CHO domains
in the engineering of protein-based sensors
within mixed C10CHO/C6 (0.02 mM, 1:1) SAMs formed from natural growth and
where a wide dynamic range of analyte bindnanografting processes. (A) 500 nm ⴛ 500 nm topographic image of the nanografted nanostructure surrounded by a mixed SAM from natural growth. (B)
ing is necessary. In comparison to prior apZoom-in AFM image showing the phase separation of the mixed SAM in the surproaches, this new method has advantages of
rounding area. (C) Zoom-in high-resolution image showing the detailed phase
spatial precision and the ability to multiplex for separation structures of the nanostructure. (D) AFM topographic image of the
same area as in (A) after LYZ injection, 5 ␮g/mL lysozyme in HEPES buffer (pH 6.8)
micro- and nanodevice applications. Further,
to the mixed SAMs for 10 min.
the strong dependence on the local structure
and environment at the nanometer level furnology with molecular precision is the right tool for
ther demonstrates the multivalent nature of protein investigation and control of protein⫺surface interattachment to surfaces. This multivalent interaction
actions. Work is in progress to explore the regulation
occurs between each protein molecule and surface
of more complex protein molecules and to further
ligands underneath, that is, in the length scale of
improve the accuracy and precision of this
several to tens of nanometers. Therefore, nanotech- regulation.
MATERIALS AND METHODS
Alkanethiol Materials. Hexanethiol (hereafter referred to as C6),
with a purity of more than 96%, was purchased from SigmaAldrich (Missouri, USA) and was used as received. Powder 11mercapto-1-undecanal disulfide [⫺S(CH2)10CHO]2 (referred to
as C10CHO due to the cleavage of the disulfide bond on gold)
was purchased from ProChimia (Gdansk, Poland).32 Ethanol solvent of 99.99% purity was purchased from Gold Shield Chemical
Co. (California, USA) and served as the solvent for all thiols.
Biotinylated Thiol, HSC8ⴚEG3ⴚbiotin. Solvents used in the syntheses were purchased in capped DriSolv bottles and used directly
without further purification and stored under argon. All glassware utilized in anhydrous conditions was flame-dried prior to
use. Glass-backed TLC plates (Silica Gel 60 with a 254 nm fluorescent indicator) were used without further manipulation and
stored over desiccant. Developed TLC plates were visualized under a short-wave UV lamp and stained with I2 and/or by heating plates that were dipped in ammonium molybdate/cerium(IV)
sulfate solution. Flash column chromatography (FCC) was performed using silica gel (32⫺63 ␮m) and employed a solvent polarity correlated with TLC mobility. NMR spectra (Varian INOVA
600 MHz, California, USA) were obtained using a 600 MHz instru-
www.acsnano.org
ARTICLE
for covalent or electrostatic immobilization should
take protein size and binding site distribution into
consideration for effective immobilization
regulation.
ment with CDCl3 as a solvent, and chemical shifts were referenced to residual CHCl3 (7.26 ppm) and CDCl3 (77.1 ppm). Highresolution mass spectra were recorded at the UC Davis Molecular
Structure Facility using MALDI-TOF (Applied Biosystems 4700
Proteomics Analyzer, California, USA) with internal calibration. Infrared spectra were acquired using an ATR-FTIR spectrometer
(Spectrum 100 FTIR spectrometer, Massachusetts, USA).
N-Boc-N=-biotinyl-3,6-dioxaoctane-1,8-diamine (3). To a solution of
mono-Boc-protected amine 2 (2.04 g, 8.2 mmol) in 80 mL of
DMF and 3.43 mL of TEA (25 mmol) stirred under argon was
added D-biotin-2,3,5,6-tetrafluorobenzoate 1 (3.21 g, 8.8 mmol).
The reaction mixture was stirred at room temperature for 24 h
and then concentrated in vacuo. The resultant crude product was
purified by FCC using a gradient of 16:1 to 8:1 CH2Cl2/MeOH to
afford 3 as a white solid. NMR and mass spectral data of 3 are
consistent with the literature.38,39
N-Biotinyl-3,6-dioxaoctane-1,8-diamine (4). Reagent K39 was prepared in a separate glass-stoppered flask and contained the following: 83% TFA, 5% phenol, 5% H2O, 5% thioanisole, 3% 1,2ethanedithiol. To 3 (1.26 g, 2.7 mmol) was added a 10 mL
solution of Reagent K, and the mixture was stirred at room temperature for 1.5 h. The reaction mixture was then concentrated
VOL. 2 ▪ NO. 11 ▪ 2374–2384 ▪ 2008
2381
ARTICLE
Scheme 1. Synthesis Route of N-Biotinyl-N=-(11-mercaptoundecyl)-3,6-dioxaoctane-1,8-diamine
and azeotroped several times with toluene. The crude product
was purified by FCC using a gradient of 10:1 to 5:1 CH2Cl2/MeOH
with 1% TEA added to afford 4 as brown oil. NMR and mass spectral data of 4 are consistent with the literature.38,39
N-Biotinyl-N=-(11-mercaptoundecyl)-3,6-dioxaoctane-1,8-diamine (6). A
solution of 11-mercaptoundecanoic acid 5 (260 mg, 1.2 mmol)
in anhydrous CH2Cl2 (20 mL) was added to a suspension of PScarbodiimide (1.42 g, 1.9 mmol) in 45 mL of anhydrous CH2Cl2
that was stirred for 5 min. After 5 min, a solution of 4 (353 mg,
0.94 mmol) in a 1:1 mixture of anhydrous DMF and pyridine (35
mL) was cannulated into the suspension. The reaction mixture
was allowed to stir for 18 h at room temperature under argon.
The resin was filtered off and washed with MeOH and CH2Cl2.
The filtrate was concentrated, and the resulting material was purified by FCC using 10:1 CH2Cl2/MeOH to afford 3 (304 mg, 0.5
mmol) as a white solid in 56% yield (Scheme 1): 1H NMR (600
MHz, CDCl3) ␦ 6.49 (t, J ⫽ 5.7 Hz, 1H), 6.31 (t, J ⫽ 5.1 Hz, 1H),
6.26 (s, 1H), 5.25 (s, 1H), 4.52⫺4.50 (m, 1H), 4.33⫺4.31 (m, 1H),
3.62 (s, 4H), 3.57 (app t, J ⫽ 5.4 Hz, 4H), 3.50⫺3.39 (m, 4H),
3.17⫺3.14 (m, 1H), 2.92 (dd, J ⫽ 5.1, 12.9 Hz, 1H), 2.74 (d, J ⫽
13.2 Hz, 1H), 2.52 (app q, J ⫽ 7.2 Hz, 2H), 2.23 (t, J ⫽ 7.5 Hz, 2H),
2.18 (t, J ⫽ 7.5 Hz, 2H), 1.75⫺1.57 (m, 12H), 1.23 (br s, 11H); 13C
NMR (150 MHz, CDCl3) ␦ 173.6, 173.3, 163.8, 70.17, 70.14, 70.13,
70.0, 61.8, 60.2, 55.5, 40.6, 39.23, 39.20, 36.7, 36.0, 34.1, 29.55,
29.52, 29.47, 29.42, 29.1, 28.4, 28.16, 28.13, 25.8, 25.6, 24.7;
MALDI-TOF-MS calcd for C27H50N4O5S2 [M ⫹ H]⫹ 575.32, found
575.35, [M ⫹ Na]⫹ 597.32, found 597.33; FTIR 1552 (amide II
band) 1643 (CAO amide, str), 1702 (CAO urea, str), 2852, 2922,
3287 (N⫺H str).40,41
Preparation of Self-Assembled Monolayers. The SAM preparation
used in this study follows established procedures.34,42 Gold
(99,999%, Alfa Aesar, Massachusetts, USA) was deposited in a
high-vacuum evaporator (Model DV502-A, Denton Vacuum, New
Jersey, USA) at a base pressure below 2 ⫻ 10⫺7 Torr onto freshly
cleaved mica substrates (clear ruby muscovite, S&J Trading Co.,
New York, USA). The mica was preheated and maintained at 350
°C before deposition using two quartz lamps mounted behind
the mica. The substrate heating led to the formation of relatively
large Au(111) terraces. Typical evaporation rates were 0.3 nm/s,
and the thickness of the gold films ranged from 150 to 200 nm.
After the evaporation, the gold thin films were annealed at 350
°C under vacuum for 30 min and allowed to cool to room temperature. The gold films were then transferred into the corresponding alkanethiol solutions within 5 min after removed from
the vacuum chamber to avoid contamination. The two mixed
C10CHO/C6 SAMs were formed in solutions containing an equal
molar ratio and a total concentration of 0.1 and 0.02 mM, respectively. Biotin-terminated thiol SAMs were formed by immersing
a gold film in a 0.1 mM solution for 48 h.
2382
VOL. 2 ▪ NO. 11 ▪ TAN ET AL.
Preparation of Protein Solutions. LYZ (from hen egg, 95% purity),
rabbit IgG (from rabbit serum, 95% purity), and goat antibiotin
IgG (96% purity) were purchased from Sigma-Aldrich (Missouri,
USA) and used as received. The protein solutions, such as rabbit
IgG (10 ␮g/ml) and LYZ (5 ␮g/ml), were prepared in HEPES buffer
(pH 6.8, 10 mM, Sigma-Aldrich, Missouri, USA). The goat antibiotin IgG was diluted to the desired concentration of 10 ␮g/mL
using 1⫻ PBS (pH 7.0, Sigma-Aldrich, Missouri, USA) before the
immobilization process.
For the protein immobilization on surfaces, the concentrations of 10 ␮g/mL for IgG and 5 ␮g/mL for LYS were used to
overwhelm the surface adhesion components in solution. Further, a CHO-terminated SAM was characterized in HEPES buffer
(pH 6.8) without protein in situ as a control experiment. Little adsorption was observed in this blank test to confirm that the nature of the bright spots in the AFM topographs is due to protein
attachment.
Atomic Force Microscopy Imaging and Analysis. The AFM utilized
was a home-constructed, deflection-type scanning head that exhibits high mechanical stability. The scanner was controlled by
an AFM 100 preamplifier and a STM 1000 electronics (RHK Technology, Inc. Michigan, USA). The AFM scanner was calibrated laterally via the periodicity of a mica(0001) surface (0.518 nm) and
vertically using single atomic steps of a Au(111) (0.235 nm).
Sharpened Si3N4 microlevers (Veeco Metrology Group, California, USA) with a force constant of 0.1 N/m were used for AFM imaging. Images were acquired using contact mode in specified liquid media. The typical imaging force is approximately 5 nN.
Both domain size and domain spacing were measured quantitatively from more than 30 cursor profiles per image to get sufficient statistics. In mixed component SAMs, the C10CHO domain
sizes were measured from the full width at half-maximum
(fwhm) of the peaks, and the domain separations (center-tocenter and edge-to-edge) were obtained from the separation
distances between the peaks. In the array of biotin nanostructures, the feature size and edge-to-edge separation distances
were measured from the fwhm.
For arrays 1, 2, 3, and 6, two additional experiments were
completed in addition to the data reported here. For arrays 4
and 5, three more experiments were done to ensure
reproducibility.
Acknowledgment. We thank Jessica Koehne and Susan G.
Stagner at University of California, Davis, for helpful discussions.
This work was supported by University of California, Davis; UC
Discovery Grant in conjunction with Novartis; NIH (R21
GM077850-01) and NSF (CHE-0809977). Y.H.T. is a recipient of
the Summer Graduate Student Researcher Award from University of California, Davis.
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